Fig. 2

Ajuba Colocalizes with Isl1 and Represses Its Transcriptional Activity(A) Subcellular localization of Isl1 and Ajuba in HEK293T cells. FLAG-HA-tagged Ajuba and Isl1 full-length or deletion constructs were transiently expressed and detected by immunofluorescence using anti-HA or anti-Isl1 antibodies. Scale bars, 50 μm.(B and C) HEK293T (B) and NIH 3T3 (C) cells were transiently transfected with a 40 ng (B) or 50 ng (C) luciferase reporter construct containing the Mef2c cardiac-specific enhancer fragment (Dodou et al., 2004) in front of a minimal fos promoter (Mef2c-luc), alone or together with Isl1 (400 ng), Isl1 lacking the DNA-binding domain (Isl1ΔHOMEO, 400 ng), Ajuba (400 ng), or with constant amount of Isl1 (400 ng), and increasing amounts of Ajuba expression plasmid (100, 200, 300, 400 ng). The luciferase levels were normalized for the β-galactosidase activity of a cotransfected RSV-lacZ reporter and presented as fold activation relative to the luciferase levels of the Mef2c reporter construct alone. Transfections were performed at least three times in triplicates, and representative experiments with the SDs are shown.(D) ChIP of nuclear extracts from day 5 EBs, using mouse or rabbit IgG (mIgG and rIgG, respectively) as a control, anti-Isl1 and anti-Ajuba antibodies, respectively. PCRs were performed using primers flanking the two conserved Isl1-binding sites in the Mef2c AHF enhancer.(E) Confocal images of cryosections of E9 embryos stained with anti-Ajuba (green) and anti-Isl1 (red) antibodies. Scale bars, 100 μm. See also Figure S1.